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A 532 nm red pigment formed in the thiobarbituric acid (TBA) assay of human urine was characterized after separation of the pigment by high-performance liquid chromatography. The yield of the red pigment was somewhat higher at pH 2 than at pH 5; its development was not inhibited by ethylenediaminetetraacetic acid. The characteristics of the pigment were similar to those of the pigment derived from standard malonaldehyde. The amount of the pigment formed was roughly equal to the content of malonaldehyde derivatives estimated as 1-(2,4-dinitrophenyl)pyrazole. Pigment formation was significantly enhanced by t-butyl hydroperoxide (t-BuOOH) and ferric ions, which may be due to pigment formed from aldehydes other than malonaldehyde; the presence of these aldehydes was confirmed by the formation of the corresponding 2,4-dinitrophenylhydrazones. The amount of pigment produced from 24-h urine samples of 12 healthy subjects was estimated to be 26-95 nmol/kg, and 65-182 nmol/kg in the presence of t-BuOOH. These values are lower than those for urine of rabbit or rat. The TBA reactivity in the absence and presence of t-BuOOH of human urine was not related to age or sex. The TBA reactivity of human urine collected in the afternoon and in the evening was higher than that of urine collected in the morning.

Detection and quantitative determination of the components : i) radical species, ii) hydroperoxides, iii) secondary aldehydes, hydrocarbons and fatty acids, and iv) reaction products between proteins, which are generated during lipid peroxidation, are reviewed. Determination of plural classes of components is required for the accurate monitoring of lipid peroxidation. Among the many known assay methods, thiobarbituric acid test can be properly used for the monitoring of lipid peroxidation. The principles of the test, which has been recently clarified, is reviewed in detail.

The thiobarbituric acid (TBA) reactivity of oxidized methyl linoleate, soybean oil, sesame oil, lard, chicken oil and sardine oil was characterized by using four different methods with 0.01% butylated hydroxytoluene (BHT). Optimal pH for the reactivity of most of the oxidized samples was 3–4, and that of some samples was above 5. Introduction of 2 mMt-butyl hydroperoxide (t-Bu00H) or 0.2 mM ferric ion in the reaction markedly enhanced the reactivity. Introduction of 0.2 mM ethylenediamine tetraacetic acid suppressed the reactivity. The characteristics of the TBA-reactivity of the samples were similar to those of alkadienals or alkenals. The most preferable method for the estimation of the TBA-reactive substances of the oxidized fats and oils was that using solvents at pH 3.5 with introduction of BHT, andt-Bu00H or ferric ion.

In the thiobarbituric acid (TBA) assay of oxidized oils, the development of red 2 : 1 TBA-malonaldehyde adduct was enhanced by ferric and ferrous ions and suppressed by EDTA in a dose-dependent manner. The effect of the ions could not be ascribed to the increased release of malonaldehyde from the oils, but was reasonably ascribable to the increased TBA-reactivity of alkadienals and/or alkenals released from the oils, since the reactivity of these aldehydes was quite similarly enhanced by the ions and suppressed by EDTA. The development of the adduct from the oils, alkadienals and alkenals was suppressed by cupric ion, probably owing to its activity to destroy the adduct. These findings strongly support the idea that alkadienals and/or alkenals are the major TBA-reactive substances in the oxidized oils.

The level of urinary thiobarbituric acid (TBA) reactants in healthy human subjects due to malonaldehyde derivatives was measured to assess the lipid peroxidation status of the whole body. For each subject the TBA reactant level over a day varied over a 2-3 fold range while the daily level varied over a 1.5-3 fold range under normal life-style conditions. One of the factors causing an increase in the reactant level within a single day may be the subjects's physical activity, because the reactant level of each subject was higher in the afternoon or in the evening than in the morning. Remaining awake all night or hard exercise caused a dramatic increase in the reactant level over a day and in the daily reactant level. The reactant level within a single day for a subject was increased 5.5 fold and the daily level 3 fold by remaining awake all night, and the level within a day was increased 22 fold by hard exercise while the corresponding daily level was increased 7 fold. It is unlikely that food, alcohol and smoking greatly affect the reactant level. The results suggest that increased physical activity enhances lipid peroxidation in the whole body and thus the increased urinary excretion of malonaldehyde derivatives.

The thiobarbituric acid (TBA) reactivity of rat liver and brain homogenates was characterized to elucidate what kinds of aldehyde species contributed to the reactivity. Characteristic pH dependence of the reactivity with a maximum at around pH 3 and marked enhancement of the reactivity by t-butyl hydroperoxide (t-BuOOH) and ferric ion were similar to those of alkadienals. The amounts of aldehyde species, including alkadienals determined as 2,4-dinitrophenylhydrazones, were high enough to account for the enhanced reactivity. The reactivity was inhibited by ethylenediaminetetraacetic acid (EDTA) but not completely, suggesting the presence of malonaldehyde whose reactivity was not affected by EDTA. The amounts of malonaldehyde determined as 1-(2,4-dinitrophenyl)pyrazole could account for a part of the reactivity in the presence of EDTA. Hence, the TBA reactivity of liver and brain homogenates at around pH 3 in the presence of t-BuOOH and ferric ion may be accounted for by alkadienals and malonaldehyde and that in the presence of EDTA by malonaldehyde.

The fluorometric thiobarbituric acid (TBA) assay of blood plasma was performed under various conditions in order to assess whether the assay reflects lipid peroxidation. The TBA-reactivity of malonaldehyde was not dependent on the pH values of the reaction and was little affected by tert-butyl hydroperoxide (tert-BuOOH). The rectivity of 2, 4-nonadienal and 2-hexenal, which was maximal at pH 3-4 and at above pH 4, respectively, was dramatically enhanced by tert-BuOOH and ferric ion, and suppressed by ethylenediaminetetraacetic acid (EDTA). The pigment formation from oxidized low-density-lipoprotein was maximal at about pH 5, markedly enhanced by ferric ion and suppressed by EDTA, indicating that the TBA-reactive substances in the oxidized lipoprotein were composed of malonaldehyde, alkadienals and alkenals originated from lipid peroxidation. The pigment formation from plasma and its phosphotungstic acid precipitate was maximal at pH 4 and at below pH 2, respectively. The effect of tert-BuOOH, ferric ion and EDTA was not significant. The major TBA-reactive substances from plasma may be compounds other than malonaldehyde, alkadienals, alkenals and those in the oxidized lipoprotein. The TBA-reactivity of plasma does not appear to be specific to lipid peroxidation.

Thiobarbituric acid (TBA) assay of rat liver homogenate was performed by four general variations with 0.01% butylated hydroxytoluene. Development of red pigment was greatly dependent on the methods. The pigment formation by each method was dramatically increased by introduction of 2 mM t-butyl hydroperoxide (t-BuOOH). The pH values of the reaction mixtures greatly affected the pigment yield both in the absence and presence of t-BuOOH, and the maximal pigment yield was obtained at pH 3-4. These characteristic profiles of the pigment formation were similar to those from alkadienals and essentially different from those from malonaldehyde. Alkadienals were likely candidates for the TBA-reactive substances in the homogenate.

Thiobarbituric acid (TBA) reaction of several aldehydes and oxidized lipids in glacial acetic acid was performed. All the samples were freely soluble in the solvent used. Saturated aldehydes produced a stable yellow pigment with an absorption maximum at 455 nm, a red pigment derived from malonaldehyde at 532 nm, and an orange pigment due to dienals at 495 nm. The absorbance maximum was 7–9 per μmol for saturated aldehydes, 27.5 per μmol for malonaldehyde and about 2 per μmol for dienals. Autoxidation of unoxidized lipids increased progressively in glacial acetic acid. When the TBA test was performed under nitrogen, autoxidation of unoxidized lipids was inhibited completely. While saturated aldehydes produced no yellow pigment under nitrogen, oxidized lipids produced a considerable amount of stable yellow pigment. The value for absorbance at 455 nm as a function of autoxidation time paralleled those of peroxide values. The absorbance of most oxidized lipids at 455 nm was higher than at 532 nm. Yellow pigment formation in the TBA test under nitrogen could not be ascribed to free saturated aldehydes but rather to unspecified closely related substances. The stable yellow pigment was found to be an excellent indicator of lipid oxidation.

A mixture of malonaldehyde, an alkenal, an alkadienal and an organic hydroperoxide, as an oxidized lipid model, was made to react with thiobarbituric acid (TBA) in 4% acetic acid at 100°C for 20min. Amount of a red pigment, formed from the alkenal and the alkadienal, was greatly increased with the amounts of other components. A combination of that alkenal and alkadienal or that of two alkadienals synergistically enhanced the pigment formation. Organic hydroperoxides dramatically enhanced this formation from these aldehydes. Intermediary 1:1 adducts of the aldehydes with TBA were effectively converted to the pigment in the presence of hydroperoxides. Pigment yield was much greater than that of the malonaldehyde content in the mixture. Alkenals, alkadienals and hydroperoxides may contribute to formation of red pigment synergistically in TBA tests for oxidized lipids.

The reaction of thiobarbituric acid (TBA) with saturated aldehydes, i.e., 1-butanal, 1-hexanal and 1-heptanal, produced a 455-nm yellow and a 532-nm red pigment. Formation of the pigments depended on the reaction conditions. The yellow pigment was unstable in the presence of excess amounts of the saturated aldehydes. The red pigment was formed only when the reaction was performed at a TBA/aldehyde ratio of 1:1 in aqueous acetic acid. Formation of the yellow and red pigments required molecular oxygen. The colorless adducts, intermediates for the yellow and the red pigments, were isolated from the reaction mixtures. Aldol condensation and dehydration of 2 mol of the saturated aldehydes initially gave the alpha, beta-unsaturated aldehydes, which in turn reacted with TBA to form the colorless adducts, pyranopyrimidine derivatives. The adducts were then converted into the yellow and red pigments under aerobic conditions.

The thiobarbituric acid (TBA) reaction was performed on linoleic acid 13-monohydroperoxide, autoxidized fatty esters, edible fats and oils, rat liver microsomal lipids, and on human erythrocyte ghost lipids in order to determine which substances from peroxidized lipids are TBA-reactive. The reaction was carried out in 2% acetic acid containing butylated hydroxytoluene using two different reaction modes: a one-step mode which involves heating at 100°C, and a two-step mode which involves first treatment at 5°C and subsequent heating at 100°C. Yields of the red 1∶2 malonaldehyde/TBA adduct, as estimated by absorbance, fluorescence intensity and high-performance liquid chromatography, were much higher than the malonaldehyde content as determined by direct chemical analysis. Yields of red pigment obtained by the two-step mode were slightly higher than those obtained by the one-step mode. Pigment yields were dramatically increased by addition oft-butyl hydroperoxide. Red pigment formation from alkenals and alkadienals was similarly enhanced by the two-step mode or by addition oft-butyl hydroperoxide, whereas pigment formation from malonaldehyde was not. It appears likely that a component of the total red pigment formed from the peroxidized lipids was due to aldehyde species other than malonaldehyde.

This paper describes what components produce a red pigment in the thiobarbituric acid (TBA) test of peroxidized lipids. Alk-2-enals, alka-2,4-dienals and hydroperoxide functions are potential candidates for the red pigment in the TBA test. The TBA test can be regarded as an excellent method to reflect the combined effect of these components and thus the degree of lipid oxidation.

Malondialdehyde (MDA) was found to react with normal hemoglobin A (Hb A), forming a number of less cationic components which were detected by cellulose acetate electrophoresis and gel electrofocusing. All the modified components moved down the cation-exchange resin at a quicker rate than Hb A, and this chromatographic behavior of the modified components was similar to that of glycosylated Hb A. Some of these modified components were intermolecularly crosslinked, and showed fluorescence with an excitation maximum at 390 nm and an emission maximum at 460 nm. It is likely that MDA reacts nonspecifically with the epsilon-amino groups of lysine and N-terminal amino groups to produce aminoacrolein, crosslinks, and strongly fluorescent 1,4-dihydropyridine-3,5-dicarbaldehyde. Oxygen affinity of the modified hemoglobins was increased. The modified hemoglobins were more readily oxidized into met-form. Mechanical stability of Hb A was also decreased by the modification. These results suggest that a considerable conformational change in Hb A was induced by the treatment with MDA. Since MDA is generated in erythrocytes as a consequence of liquid peroxidation, MDA may react with intracellular Hb A and influence the function and the stability of hemoglobin.

The reaction of four to eight carbon straight-chain alk-2-enals with 2-thiobarbituric acid (TBA) produced yellow 455-nm-, orange 495-nm-, and red 532-nm-absorbing pigments depending upon the reaction conditions. The 1:1 reaction of the aldehydes with TBA in 15% acetic acid at 100 degrees C produced the yellow pigment at 0.25 h and the red at 6 h. The reaction of the aldehydes with TBA in excess at 100 degrees C produced the yellow at 0.25 h, the orange at 2-6 h, and the red at 0.25-6 h. The formation of these pigments required molecular oxygen. These pigments could be separated from each other on HPLC. The red pigment formed from the aldehydes could not be distinguished from the red 1:2 malonaldehyde-TBA adduct by absorption spectrum and HPLC. The red color yield was the highest in the 1:1 reaction and retarded in the reaction with TBA in excess. The red color due to these aldehydes may contribute in part to the color formed in the general TBA test of lipid oxidation. The 1:1 reaction initially produced colorless 1:1 adducts X, which were subsequently converted into the yellow and red pigments under aerobic conditions. The reaction of the aldehydes with TBA in excess might initially produce X and then another colorless 1:2 adducts Y; the latter being converted into yellow, orange, and red pigments under aerobic conditions.

When autoxidized chicken fat and unsaturated fatty acids were subjected to thiobarbituric acid (TBA) test in trichloracetic acid, all samples revealed two absorption maxima at 532 and 455 nm. Rate of increase in the TBA-reactive substances (TBARS) at 532 nm was different from that in the TBARS at 455 nm. It was suggested that there were several kinds of TBARS in the autoxidized samples: the TBARS at 532 mn which were unstable in the autoxidation process and slowly liberated malonaldehyde; the TBARS at 532 nm which liberated malonaldehyde rapidly; and the TBARS at 455 nm which liberated other aldehydes unstable after reaction with TBA.